We propose to develop a high throughput in vitro assay to identify genetic factors affecting toxicant impact on neuronal differentiation. This project uses a novel genetic mapping resource developed at Predictive Biology, the Diversity Outbred (DO) Mouse ES Cell Panel. Using robust ES cell engineering methods, we will incorporate neuronal differentiation reporters into DO ES lines. Neuronal Stem Cell (NSC) lines will be derived from the panel of modified DO ES lines, and the NSC panel will be used to quantify neuronal differentiation after toxicant exposure. These data will be used to map loci and identify genes that mediate variable response to neurotoxicants. The project design prioritizes rapid, sensitive and highly scalable assay technology, and features a cassette approach to vector design to facilitate construction of multiple assay panels to track toxicant effects on specific types or classes of neurons and neuronal cells. This technology will enable primary screening of large numbers of compounds at HTS scale, and will be useful for prioritizing compounds for follow up studies in vitro and in vivo using both mouse and human cells as well as live animal models. The genes and toxicological pathways identified using this technology will ultimately improve human health by providing a more detailed mechanistic understanding of human developmental neurotoxicity, and will contribute to a genetically informed approach to estimating risk associated with exposure to neurotoxicants. !